Abstract
Citrobacter freundii is a nosocomial pathogen increasingly associated with multidrug resistance and hospital outbreaks. Despite its growing clinical relevance, no standardized core genome multilocus sequence typing (cgMLST) scheme has been available for high-resolution epidemiological analyses. Here, we developed and validated a robust cgMLST scheme for C. freundii comprising 3,250 target loci based on a curated data set of 825 globally distributed genomes representing extensive sequence type and geographic diversity. Validation against published outbreak data sets from hospitals in Finland and Belgium, as well as environmental and patient isolates from two German university hospitals, proved that the scheme possesses high target gene coverage (median 99.6%) and strong discriminatory power. Additionally, we developed a combined cgMLST scheme for C. freundii, C. portucalensis, C. braakii, and C. europaeus, based on 2,307 shared target loci and target gene coverages of ≥99.7%. This scheme proved suitable for cross-species outbreak analysis. Our analyses revealed environments, such as sinks, shower drains, and toilets, as likely reservoirs where Citrobacter species may persist in hospital settings. These findings suggest that environmental sources could play a significant role in transmission events involving patients, while allele-based cluster analyses indicated that direct patient-to-patient transmission was rare. Given the increasing prevalence of multidrug-resistant (MDR) Citrobacter strains, the level of discrimination achieved by these newly developed cgMLST schemes underscores the importance of accurate species identification and environmental screening in understanding the transmission dynamics of opportunistic healthcare-associated pathogens. Ultimately, this makes them valuable tools for genomic surveillance, outbreak investigation, and infection prevention. IMPORTANCE: Accurate identification and high-resolution typing of multidrug-resistant bacteria are essential for understanding their transmission dynamics in hospitals, particularly in light of the global spread of resistant strains and the role of environmental reservoirs. The newly developed cgMLST schemes presented here provide standardized, portable tools for both local and global scientific and clinical communities to conduct fine-scale genomic epidemiology across four Citrobacter species. These schemes support detailed outbreak reconstruction, source attribution, and cross-hospital comparisons, capabilities that are critical in an era of increasing antimicrobial resistance and international patient movement. By enabling consistent, species-specific surveillance and comparative analyses, cgMLST enhances infection control and public health responses, facilitating early detection and targeted intervention.